This invention relates to the field of immunoglobulin production and modifications to naturally occurring antibody amino acid sequences. Specifically, the invention relates to using recombinant DNA techniques to produce chimeric genes and to take advantage of these gene modification techniques to construct chimeric antibodies.
Antibodies are specific immunoglobulin (Ig) polypeptides produced by the vertebrate immune system in response to challenges by foreign proteins, glycoproteins, cells, or other antigenic foreign substances. The sequence of events which permits the organism to overcome invasion by foreign cells or to rid the system of foreign substances is at least partially understood. An important part of this process is the manufacture of antibodies which bind specifically to a particular foreign substance. The binding specificity of such polypeptides to a particular antigen is highly refined, and the multitude of specificities capable of being generated by the individual vertebrate is remarkable in its complexity and variability. Millions of antigens are capable of eliciting antibody responses, each antibody almost exclusively directed to the particular antigen which elicited it.
Two major sources of vertebrate antibodies are presently utilizedxe2x80x94generation in situ by the mammalian B lymphocytes, and generation in cell culture by B-cell hybrids. Antibodies are generated in situ as a result of the differentiation of immature B lymphocytes into plasma cells, which occurs in response to stimulation by specific antigens. In the undifferentiated B cells, the portions of DNA coding for the various regions on the immunoglobulin chains are separated in the genomic DNA. The sequences are assembled sequentially prior to expression. A review of this process has been given by Gough, Trends in Biochem. Sci., 6:203 (1981).
The resulting rearranged gene is capable of expression in the mature B lymphocyte to produce the desired antibody. However, even when a particular mammal is exposed to only a single antigen a uniform population of antibodies does not result. The in situ immune response to any particular antigen is defined by the mosaic of responses to the various determinants which are present on the antigen. Each subset of homologous antibodies is contributed by a single population of B cellsxe2x80x94hence in situ generation of antibodies is xe2x80x9cpolyclonalxe2x80x9d.
This limited but inherent heterogeneity has been overcome in numerous particular cases by use of hybridoma technology to create xe2x80x9cmonoclonalxe2x80x9d antibodies in cell cultures by B cell hybridomas [See Kohler and Milstein, Nature 256:495-497 (1975)].
In this process, the relatively short-lived, or mortal, splenocytes or lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line, thus producing hybrid cells or xe2x80x9chybridomasxe2x80x9d which are both immortal and capable of producing the genetically coded antibody of the B cell. The hybrids thus formed are segregated into single genetic strains by selection, dilution, and regrowth, and each strain thus represents a single genetic line. They therefore, produce antibodies which are assured to be homogeneous against a desired antigen. These antibodies, referencing their pure genetic parentage, are called xe2x80x9cmonoclonalxe2x80x9d.
Monoclonal antibodies with mono-specificity have greatly influenced immunology, and their usefulness has already been demonstrated in such sciences as biology, pharmacology, chemistry and others. Such monoclonal antibodies have found widespread use not only as diagnostics reagents [see, for example, Immunology for the 80""s, Eds. Voller, et al., MTP Press, Lancaster, (1981), but also therapy (see, for example, Ritz and Schlossman, Blood, 59:1-11, (1982)].
Monoclonal antibodies produced by hybridomas, while theoretically effective as discussed above and clearly preferable to polyclonal antibodies because of their specificity, suffer from an important disadvantage. In many applications, the use of monoclonal antibodies produced in non-human animals is severely restricted where the monoclonal antibodies are to be used in humans. Repeated injections of a xe2x80x9cforeignxe2x80x9d antibody in humans, such as a mouse antibody, may lead to harmful hypersensitivity reactions. Such a non-human derived monoclonal antibody, when injected into humans, causes a anti-nonhuman antibody (ANHA) response. For a discussion of a specific ANHA response caused by using murine-derived antibodies, human anti-mouse antibody (HAMA) response, see Shawler et al., Journal of Immunology, 135:1530-1535 (1985).
It is believed that animal immunoglobulins having human constant regions will generate less of an ANHA response when injected into humans than animal immunoglobulins having nonhuman constant regions. As such, monoclonal antibodies having good binding affinities for selected antigens and having human constant regions are thought to possess great potential utility for immunological diagnosis and therapy of human patients with cancer.
Various attempts have so far been made to manufacture human-derived monoclonal antibodies by using human hybridomas. For example, human-human hybridomas [Olsson et al., Proc. Natl. Acad. Sci. (USA), 77:5429 (1980)]; human-murine hybridomas [Schlom et al., Proc. Natl. Acad. Sci. (USA), 77:6841 (1980)] and several other xenogenic hybrid combinations have been prepared. Human monoclonal antibodies have also been produced by transformation of lymphocytes using Epstein-Barr virus. However, such hybridomas may potentially harbor pathogenic human viruses. Alternatively, primary, antibody producing B cells have been immortalized in vitro by transformation with viral DNA. Unfortunately, yields of monoclonal antibodies from human hybridoma cell lines are relatively low (1 xcexcg/mL in human compared to 100 xcexcg/mL in mouse hybridomas), and production costs are high.
While human immunoglobulins are highly desirable in immunological diagnosis and therapy of human cancer patients, human hybridoma techniques have not yet reached the stage where human monoclonal antibodies with required antigenic specificities can be easily obtained. In addition, for obvious ethical reasons, researchers can not immunize human subjects with selected toxic or otherwise deleterious antigens to generate antibodies against the specific antigen. This imposes great restrictions on immunological diagnosis and therapy of human patients.
No human antibody has been isolated which relatively strongly binds to TAG-72. Consequently, suitable antibodies must be engineered. The production of human-derived monoclonal antibodies is certainly possible, but is still inefficient in view of its low reproducibility and the other problems noted above. Consequently, most monoclonal antibodies are derived from non-human animals.
A monoclonal antibody which reacts with high binding affinity to human tumor antigens, but which is not recognized as a foreign substance by humans is highly desirable. A method to overcome this difficulty is to create artificially, an antibody which is very similar to a human antibody and is not recognized as a foreign substance within the human body, i.e., a chimeric, or xe2x80x9chumanizedxe2x80x9d antibody.
Typically in chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from humans. One clear advantage to such chimeric forms is that, for example, the variable regions can conveniently be derived from presently known sources using readily available hybridomas of B cells from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the specificity of the variable region is not affected by its source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non-human source.
One known human tumor antigen is tumor-associated glycoprotein (TAG-72). TAG-72 is associated with the surface of certain tumor cells of human origin, specifically the LS174T tumor cell line. LS174T [American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 (herein ATCC) No. CL 188] is a variant of the LS180 (ATCC No. CL 187) colon adenocarcinoma line.
The karyotype of LS174T is similar to that of LS180 with a missing X chromosome in a majority of the cells. Data has been presented as described in Johnson, et al., Cancer Res., 46:850-857 (1986), to characterize the TAG-72 molecule as a mucin. This conclusion is based on the following observations: (a) TAG-72 has a high molecular weight ( greater than 1xc3x97106) as shown by its exclusion from a SEPHAROSE CL-4B column (modified polysaccharide gel filtration column from Pharmacia, Inc., Piscataway, N.J.); (b) the density of TAG-72 determined by equilibrium centrifugation in CsCl was 1.45 gm/mL, indicating a heavily glycosylated glycoprotein; (c) TAG-72 demonstrates a change in migration after neuraminidase digestion, indicating that it is a heavily sialylated molecule with an abundance of O-glycosidically linked oligosaccharides characteristic of mucins; (d) blood group antigens commonly found on mucins are found on affinity-purified TAG-72; and (e) Chondroitinase ABC digestion had no effect on TAG-72, thus demonstrating that the TAG-72 epitope is not expressed on a chondroitin sulfate proteoglycan.
Numerous murine monoclonal antibodies have been developed which have binding specificity for TAG-72. One of these monoclonal antibodies, designated B72.3, is a murine IgGl produced by hybridoma B72.3 (ATCC No. HB-8108). B72.3 is a first generation monoclonal antibody developed using a human breast carcinoma extract as the immunogen (see Colcher et al., Proc. Natl. Acad. Sci. (USA), 78:3199-3203 (1981); and U.S. Pat. Nos. 4,522,918 and 4,612,282). As used herein, the expression xe2x80x9cfirst generation monoclonal antibodyxe2x80x9d means a monoclonal antibody produced using, as the immunogen, a crude cell extract.
Other monoclonal antibodies directed against TAG-72 are designated xe2x80x9cCCxe2x80x9d (colon cancer). CC monoclonal antibodies are a family of second generation murine monoclonal antibodies. As used herein, the expression xe2x80x9csecond generation monoclonal antibodyxe2x80x9d means a monoclonal antibody produced using, as the immunogen, an antigen purified with a first generation monoclonal antibody. CC monoclonal antibodies were prepared using TAG-72 purified with B72.3. A discussion of the method for producing the CC antibodies is set forth in U.S. Pat. No. 7,073,685 (U.S. Pat. No. 7,073,685, abandoned, continued as U.S. Pat. No. 7,547,336, now U.S. Pat. No. 5,512,443), the application was filed by Schlom et al. on Jul. 15, 1987 and is available to the public from the National Technical Information Service. Because of their relatively good binding affinities to TAG-72, the following CC antibodies have been deposited at the ATCC, with restricted access having been requested: CC49 (ATCC No. HB 9459); CC 83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9458); CC92 (ATCC No. HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No. HB 9455); and CC15 (ATCC No. HB 9460).
U.S. Pat. No. 7,073,685 teaches that the CC antibodies may be altered into their chimeric form by substituting, e.g., human constant regions (Fc) domains for mouse constant regions by recombinant DNA techniques known in the art. It is believed that the proposals set out in U.S. Pat. No. 7,073,685 did not lead to an actual attempt to express any chimeric Ig polypeptide chains, nor to produce Ig activity, nor to secrete and assemble Ig chains into the desired chimeric Igs.
It is known that the function of an Ig molecule is dependent on its three dimensional structure, which in turn is dependent on its primary amino acid sequence. Thus, changing the amino acid sequence of an Ig may adversely affect its activity. Moreover, a change in the DNA sequence coding for the Ig may affect the ability of the cell containing the DNA sequence to express, secrete or assemble Ig.
Numerous articles confirm the fact that an antibody has a very complicated, a delicate three-dimensional structure. Dr. Kameyma Koh-Zoh commented, in an article in Saibo Kogaku, 4(12):1025-1035 (1985), in attempting to prepare a chimeric antibody to a melanoma antigen:
Using a chimeric antibody purified by means of HPLC, its bindability to a purified melanoma antigen was measured, but regretfully, for the time being there could not be obtained results showing its binding activity. As causes, first, there is a possibility that the C region affected the steric structure of the V region caused by the changing the mouse IgM antibody to human IgG antibody.
Idiotypes are antigenic determinants that involve variable regions of heavy and light chains of immunglobulin molecules. Isotypes and/or allotypes are antigenic determinants that are restricted to the constant regions of heavy chains. Attention is further directed to Morahan et al., Nature, 301:720-722 (1983), which teach:
The T15 idiotype, defined by sera raised in A strain mice, or in rabbits, is considered identical to that expressed by the majority of BALB/c anti-PC antibodies. To define the idiotypic determinants (idiotopes) of which the T15 idiotype is comprised, monoclonal anti-T15 antibodies were used here to examine both serum and monoclonal anti-PC antibodies. The latter were found to differ from T15 with respect to the idiotope defined by the monoclonal anti-idiotope antibody, 21A5, in that the ""21A5 idiotope"" was absent from anti-PC sera; of the monoclonal anti-PC antibodies examined, only those which were both T15+ and of the IgA isotype seemed to express this idiotype fully. This result suggests that not only the V region, but also the constant region, of the immunoglobulin molecule can contribute to the formation of an idiotypic determinant. (emphasis added)
Nishinarita et al., The Journal of Immunology, 134(4):2544-2549 (1985) teach:
Recently, Morahan et al. (12) described an anti-T15 hybridoma antibody, 21A5, that identified an idiotypic determinant associated with T15 Id and the IgA CH region . . . we have shown that NL24 binding to C3 is inhibited by not only PC-binding IgA and T15 Id+MP, but also by numerous PC-binding hybridoma proteins (HP) and the IgA fraction of normal anti-PC antibodies of BALB/c mice and presumably other strains . . . . The high frequency of C3-24 Id expression in IgA PC-binding MP and HP and in the IgA fraction if normal antibody of BALB/c mice suggest that isotype-restricted Id may not be an unusual occurrence. (emphasis added)
Clearly, based upon the teachings in the art, the influence of a homologous constant region to the three-dimensional conformation of a particular variable region is not predictable. In other words, the teachings of the prior art suggest that the binding ability of a particular antibody may be dependent upon the unique constant region associate therewith.
It is, therefore, not at all clear from the prior art that known recombinant DNA techniques will routinely produce a chimeric animal-human antibody from selected DNA sources that generate functional chimeric antibodies which bind specifically to selected human carcinomas and which reduce the initiation of ANHA side-effects when injected into humans.
Consequently, it is an object of the present invention to fuse genes coding for at least a part of an animal Ig which binds to human carcinomas expressing TAG-72 and genes coding for at least part of a human Ig. It is a further object of the invention to achieve expression of protein which can be secreted and assembled to give a functional chimeric antibody.
It is a still further object to provide an expression vector containing a DNA sequence which encodes antibodies and portions thereof which are directed against TAG-72.
It is also an object of the invention to provide cells transformed with expression vectors containing a DNA sequence which encodes antibodies and portions thereof which are directed against TAG-72.
Finally, it is an object of the present invention to provide novel antibodies for use in in vivo diagnostic assays; in vivo therapy; and radioimmunoguided surgery.
Surprisingly, the present invention is able to meet many of these above mentioned needs and provides a method for supplying the desired antibodies.
This invention concerns an antibody or antibody fragment comprising a variable region having a heavy chain (VH), said VH being encoded by a DNA sequence effectively homologous to the VHxcex1TAG germline gene (VHxcex1TAG), wherein the variable region binds to TAG-72 at least 25 percent greater than the variable region of B72.3 binds to TAG-72, with the binding affinities of the antibody and B72.3 being measured by the same technique.
This invention also concerns a DNA sequence encoding at least a portion of an antibody heavy chain, said sequence comprising a DNA sequence segment being effectively homologous to the VHxcex1TAG germline gene (VHxcex1TAG), wherein the DNA sequence segment encodes at least a portion of a heavy chain variable region (VH).
The invention includes the aforementioned antibody alone or conjugated to an imaging marker or therapeutic agent. The invention also includes a composition comprising the aforementioned antibody in unconjugated or conjugated form in a pharmaceutically acceptable, non-toxic, sterile carrier.
The invention is directed to a method for in vivo diagnosis of cancer which comprises administering to an animal a pharmaceutically effective amount of the aforementioned composition for the in situ detection of carcinoma lesions.
The invention is also directed to a method for intraoperative therapy which comprises (a) administering to an animal a pharmaceutically effective amount of the aforementioned composition, whereby the tumors are localized, and (b) excision of the localized tumors.
In other aspects, the invention is directed to expression vectors or plasmids capable of effecting the production of such aforementioned antibodies in suitable host cells. It also includes the host cells and cell cultures which result from transformation with these vectors.
The invention is also directed to a method for finding (i.e. identifying) an unknown gene or genes encoding an unknown variable region(s), said unknown variable region having binding specificity for the same antigen as does a known variable region encoded by a known gene. Generally, a detectably labeled, nucleic acid hybridization probe is prepared which comprises at least a portion, preferably about 15 nucleotides (or base pairs) or more, of the (+) and/or (xe2x88x92) strand of the 5xe2x80x2NTR of said known gene. The probe is hybridized to nucleic acid(s) including unknown V-encoding nucleic acid(s), at least partly along their 5xe2x80x2NTR(s), to form probe-polynucleotide hybrid(s). Excess un-hybridized probe is washed away, and the remaining detectably-labeled probe-polynucleotide hybrid(s) are detected and may be isolated and/or further characterized.